Nucleic acids encompass both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA, present in all nucleated cells, carries the information needed to direct the synthesis of every protein in the body. A single alteration in the correct sequence of the four DNA bases (adenine, thymine, guanine, and cytosine) may result in a defective protein. Depending upon the protein and the affected organism, the defect may range from inconsequential to life-threatening, or may be of intermediate severity. Diseases as diverse as cystic fibrosis, some types of cancer, sickle cell anemia, and atherosclerosis are known to result from specific genetic alterations.
RNA, the intermediary between DNA and protein, is the product of transcription of a DNA template. RNA assays are being performed with increasing frequency in research and clinical laboratories. This is due at least in part to the prevalence of RNA viruses such as the human immunodeficiency virus (HIV) that causes AIDS and the hepatitis C virus (HCV), and the development of drugs used in treating infections with RNA viruses.
Nucleic acid assays are routinely performed, either manually or by automated instrumentation, in numerous reference and clinical laboratories. A nucleic acid assay may be performed to detect the presence of foreign DNA or RNA, which may indicate infection with a foreign organism. For example, a variety of molecular assays are used to establish the presence and identity of nucleic acids from the human immunodeficiency virus-1 (HIV-1), Chlamydia, and other organisms causing sexually transmitted diseases.
An individual's DNA may also be analyzed to detect, treat, and in some cases prevent genetic disease. Genotype determination of genes for factor V Leiden, hereditary hemochromatosis, lipoprotein lipase mutations, and cystic fibrosis have important implications for health management. The Human Genome Project holds the promise of many more examples of medically efficacious genetic diagnostic determinations. The recent discovery of the breast cancer associated gene (BRCA-1) has highlighted both the importance of screening individuals for predisposition to a disease, and also for the attendant need for accurate, precise, reproducible, and controlled nucleic acid assays.
Laboratories that perform clinical assays must meet federal and state accrediting agencies' requirements for quality control tests in order to obtain and maintain accreditation. For example, the National Committee for Clinical Laboratory Standards (NCCLS) specifies that quality control samples must be analyzed during every batch of patient specimens analyzed. The federal Clinical Laboratory Improvement Act of 1988 (CLIA '88) mandates similar requirements, as do inspection agencies from most states. The College of American Pathologists (CAP), a non-profit peer inspection group, also requires that quality control samples be analyzed during each analytical run.
In the field of molecular pathology and genetic testing, a quality control sample includes a reference DNA or reference RNA of known sequence quality to evaluate the reliability of all steps of a test. Such reference nucleic acid is ideally as similar as possible to the test sample, and also has broad applicability to all sample preparation and test formats. Additionally, the reference nucleic acid should be easily produced, characterized, and packaged with minimal technical capability.
Materials meeting these requirements, however, are lacking in the field of molecular pathology and genetic testing. This is due in large part to the variety of different technologies and techniques currently employed for a given diagnostic determination. For example, genetic determinations currently include the use of the polymerase chain reaction (PCR), the ligase chain reaction (LCR), branched DNA, allele specific hybridization, and direct sequence determination. In addition, so-called “home brew” produced primer oligonucleotides, and isotopically labeled or non-radioisotopic based probes are used in a variety of configurations in genetic testing, but without any systematic quality control materials, and hence without any validation.
The aforementioned factors, coupled with the lability of nucleic acids, make it virtually impossible to obtain standard reagents to qualitatively and/or quantitatively assess the overall accuracy, reliability, and efficiency of the numerous manipulations performed in all phases of a laboratory assay, that is, from sample preparation through diagnostic determination. For example, one commercially available material for use as a control in a DNA assay consists of lyophilized DNA powder to be diluted and used beginning at an amplification step, which is late in the protocol and well after sample preparation. Thus, for the steps preceding amplification there is no material containing reference DNA by which the accuracy, reliability, and efficiency of these steps may be evaluated. An additional drawback in the use of this material is the apparent lack of extraneous nucleotide residues and other milieu representative of that found in normal cellular extracts.
Even a single alteration in the base sequence of a nucleic acid may have severe consequences to a patient undergoing diagnosis of a genetic disease. Because of the importance of such assays, and also because of the wide range and large numbers of molecular diagnostic assays performed, there is a great need for stable reference nucleic acids to monitor test conditions as closely as possible.
The rapid expansion of genetic-based tests has outpaced the development of appropriate reference materials traditionally used to ensure good laboratory practice. Many current quality controls rely on previously tested clinical specimens and naked DNA, and thus suffer from confidentiality issues, risk of infectious disease, inconvenience, and the inability to validate the entire clinical process. Therefore, there is a long felt need in the art for the development of effective nucleic acid testing quality controls that are safe, convenient, and can mimic a test specimen in that they withstand all steps of the nucleic acid testing. The present invention fulfills this need.